US4937193A - Process for the genetic modification of yeast - Google Patents

Process for the genetic modification of yeast Download PDF

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US4937193A
US4937193A US07/066,931 US6693187A US4937193A US 4937193 A US4937193 A US 4937193A US 6693187 A US6693187 A US 6693187A US 4937193 A US4937193 A US 4937193A
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plasmid
yeast
dna sequence
endogenous
vector
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US07/066,931
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Edward Hinchliffe
Christine J. Fleming
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Albumedix Ltd
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Delta Biotechnology Ltd
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Assigned to DELTA BIOTECHNOLOGY LIMITED, 137, HIGH ST., BURTON ON TRENT DE14 1JZ, ENGLAND A BRITISH COMPANY reassignment DELTA BIOTECHNOLOGY LIMITED, 137, HIGH ST., BURTON ON TRENT DE14 1JZ, ENGLAND A BRITISH COMPANY ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: FLEMING, CHRISTINE J., HINCHLIFFE, EDWARD
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts

Definitions

  • This invention relates to genetic engineering in brewing yeast.
  • replicating vectors that is those which are capable of mediating their own maintenance independent of the chromosomal DNA of yeast by virtue of the presence of a functional origin of DNA replication
  • ARS autonomously replicating vectors in which the "apparent" origin of replication is derived from the chromosomal DNA of yeast and
  • centromeric plasmids which carry in addition to one of the above origins of DNA replication a sequence of yeast chromosomal DNA known to harbour a centromere.
  • antibiotics for example G418 (Jiminez et.al., 1980; Webster et.al., 1983), hygromycin B (Gritz et.al., 1983), chloramphenicol (Cohen et.al., 1980) and
  • ARS plasmids are lost at a frequency greater than 10% per cell doubling (Kikuchi, 1983). In order to ensure the stable maintenance of the transformed phenotype following continuous cell proliferation, it is necessary to maintain selection for the plasmid.
  • yeast vectors which, when introduced into the recipient, enter the host chromosome by genetic recombination between homologous sequences on the vector and the chromosomal DNA.
  • yeast vectors have an extremely low efficiency of transformation, and give rise to only about 1-10 transformants per ⁇ g of DNA (Hinnen et.al., 1978; Hicks et.al., 1979).
  • This frequency can be improved by cleaving the DNA to be transformed with a restriction endonuclease which cuts in the region of DNA homology, as this creates a highly recombinogenic molecule (Hicks et.al., 1979).
  • the present invention provides a process for the transformation of yeast, in particular brewing yeast, with a 2 ⁇ m-based recombinant plasmid.
  • the transformation of brewing yeast may be facilitated by the presence of the CUP-1 gene of yeast on the plasmid, enabling selection for copper resistant transformants, but any dominant selectable marker, including antibiotic resistance, can be used, and indeed the use of a separate marker gene, as distinct from a gene coding for the peptide product of interest, may be dispensed with altogether.
  • Stable maintenance of a recombinant ⁇ gene of interest ⁇ is achieved by directing the integration of the ⁇ gene ⁇ by genetic recombination to a site within the endogenous 2 ⁇ m plasmid of brewing yeast.
  • This plasmid is present in all proprietary strains of yeast thus far investigated (Hinchliffe & Daubney, 1986).
  • the ubiquitous nature of the endogenous 2 ⁇ m plasmid implies that this plasmid is stably maintained in brewing yeast through many generations of apparently non-selective growth.
  • the integration of the ⁇ gene(s) of interest ⁇ should be directed to a site within the 2 ⁇ m plasmid which does not adversely influence the inherent stability of the endogenous 2 ⁇ m plasmid.
  • the 2 ⁇ m plasmid of yeast is a circular DNA molecule of 6318 base-pairs for which the entire nucleotide sequence has been determined (Hartley & Donelson, 1980). It is present in approximately 50-100 copies per cell in most strains of Saccharomyces cerevisiae (Clarke-Walker & Miklos, 1974), including brewing yeast (Aigle et.al., 1984; Hinchliffe & Daubney, 1986). The plasmid is inherited in a non-Mendelian fashion (Livingston, 1977), and for this reason it has been regarded as cytoplasmic in cell location.
  • Recombination between the two inverted repeats is mediated by the product of a gene entitled FLP which is present on the plasmid itself.
  • the FLP gene encodes a protein which is capable of mediating high frequency recombination between the inverted repeat region.
  • a process for the genetic modification of yeast by the incorporation into the endogenous 2 ⁇ m plasmid of yeast a DNA sequence coding for a protein or peptide of interest comprises first transforming yeast with an integration vector comprising two copies of homologous 2 ⁇ m plasmid DNA sequences positioned in direct orientation relative to one another and encompassing the said DNA sequence and then isolating, from the transformed yeast obtained, cells containing the endogenous 2 ⁇ m plasmid modified by incorporation of the said DNA sequence but not containing the vector.
  • the DNA sequence of interest may be incorporated in the integration vector via an appropriate restriction site, for example a Bam HI site or a Kpn I site, at which a DNA sequence of interest can be inserted.
  • the vector usually also comprises an extraneous DNA sequence, i.e. a sequence not necessary or over desirable for the propagation of the plasmid in yeast, but desirable for the reasons, e.g. propagation in bacteria or other non-yeast host microorganism.
  • This extraneous DNA sequence is separated from the DNA sequence coding for the protein or peptide of interest by the homologous sequence in direct orientation.
  • the extraneous DNA sequence is a DNA sequence heterologous to yeast which assists propagation of the vector in bacteria.
  • the invention also provides a 2 ⁇ m plasmid integration vector which comprises two copies of a 2 ⁇ m homologous DNA sequence in direct orientation, a DNA sequence, usually heterologous to yeast, which assists propagation of the plasmid outside yeast, and a DNA sequence coding for a heterologous protein or peptide of interest separated from the said DNA sequence assisting propagation of the plasmid by the said homologous sequences in direct orientation.
  • integration occurs via a recombination event and results in the integration of the recombinant gene (i.e. the DNA sequence of interest) to the exclusion of the remainder of the vector DNA not encompassed between the homologous DNA repeat sequences of the vector.
  • This process ensures that only the ⁇ gene(s) of interest ⁇ is stably maintained for successive generations in yeast, e.g. brewing yeast, under non-selective conditions of growth, and thereby circumvents any deleterious effects that additional DNA sequences may have upon either the technological behaviour of the yeast or the flavour and quality characteristics of the products, e.g. beer, produced by the yeast.
  • the vector upon introduction into yeast, undergoes an intramolecular recombination between the homologous DNA repeat sequences to produce two plasmid fragments, each containing one DNA sequence with homology to the endogenous 2 ⁇ m plasmid.
  • One of these fragments carries a 2 ⁇ m origin of replication which was carried by the original vector and the other carries the other DNA sequence initially contained between the repeat sequences of the vector.
  • Recombination of the latter plasmid fragment with an endogenous 2 ⁇ m plasmid of yeast at the region of homology produces a stable integrant which comprises the endogenous 2 ⁇ m plasmid of yeast and, inserted into the region of homology, the DNA sequence of interest originally contained between the two directly oriented repeats of homologous DNA of the vector.
  • the ⁇ gene(s) of interest ⁇ can be any recombinant gene, either homologous or, more usually, heterologous to yeast.
  • the process may be used, for example, to integrate the Human Serum Albumin gene stably in brewing yeast so that it is expressed from either a constitutive yeast promoter, for example the phosphoglycerate kinase promoter (PGK), or a regulated yeast promoter, for example the GAL10/CYCl hybrid promoter as described in European Pat. Application No. 86303039.1, published under No. 201239, "Fermentation with an Inducible Gene Expression System” (Delta Biotechnology Ltd.) or the GAL10/PGK promoter (PAL) described in British Pat. Application No. 8620926 filed Aug. 29th, 1986 "Yeast Promoter” (Delta Biotechnology Ltd.).
  • PGK phosphoglycerate kinase promoter
  • PAL GAL10/PGK promoter
  • genes which can be stably integrated by this system include the DEX-1 gene of Saccharomyces diastaticus which specifies the production of an extracellular glucoamylase enzyme in brewing yeast and the ⁇ -glucanase gene of Bacillus subtilis which specifies the production of an endo-1,3-1,4- ⁇ -glucanase in brewing yeast (Hinchliffe & Box, 1985). Additional genes can be first genetically modified to control the level of gene expression or to ensure that the protein whose synthesis is mediated by the gene is secreted by the brewing yeast.
  • the gene integration process of the invention ensures stable maintenance of the ⁇ gene(s) of interest ⁇ at a high copy number, under conditions of non-selective growth. This is particularly advantageous when implementing the process described in European Pat. Application No. 86303039.1 (Fermentation with an Inducible Gene Expression System), since under these circumstances the expression of the ⁇ gene(s) of interest ⁇ is regulated so that it is not expressed during the course of the primary beer fermentation, but rather induced in a post-fermentation process. By ensuring high copy numbers of the ⁇ gene(s) of interest ⁇ it is possible to achieve high level induction during the post-fermentation process, thereby enhancing the amount of heterologous protein produced.
  • Saccharomyces diastaticus DEX-1 The stable integration of the Saccharomyces diastaticus DEX-1 gene facilitates the production of beer with an enhanced attenuation limit, since the extracellular glucoamylase partially hydrolyses starch (dextrins) present in wort.
  • starch starch
  • Plasmid pEHB11 (described in European Pat. Application No. 86303039.1, publication No. 201239, Fermentation with an Inducible Gene Expression System; Delta Biotechnology Ltd.) was transformed into the brewing yeast strains NCYC 240 (ale yeast--National Collection of Yeast Cultures, Colney Lane, Norwich, United Kingdom) and BB10.2 (lager yeast--a proprietary strain of Bass yeast) by selection for copper resistant transformants as described by Hinchliffe & Daubney (1986) NCYC 240 (pEHB11) has been deposited at the National Collection of Yeast Cultures, Colney Lane, Norwich, NR4 7AU, United Kingdom on Dec. 12th, 1984 under No. NCYC 1547.
  • Transformants were verified as being copper resistant (>1 mM CuSO 4 .7H 2 O), ⁇ -galactosidase positive (blue/green colour on M63, 2% w/v galactose and X-gal, European Pat. Application No. 86303039.1) and ⁇ -lactamase positive (Chevallier & Aigle, 1979). Transformants were grown in NEP, supplemented with 2% w/v glucose and 0.2mM CuSO 4 .7H 2 O to late stationary phase prior to sub-culturing in non-selective medium (NEP, 2% w/v glucose lacking CuSO 4 .7H 2 O).
  • NEP non-selective medium
  • yeast were harvested and plated for single colonies on NEP, 2% w/v glucose agar medium. Single colonies were then checked for the phenotypic presence of the plasmid pEHB11 by replica plating to the same medium supplemented with 0.2 mM CuSO 4 .7H 2 O and 1mM CuSO 4 .7H 2 O, as well as M63 medium supplemented with 2.0% w/v galactose and X-gal.
  • Copper resistant ⁇ -galactosidase negative cells were subjected to a full molecular biological analysis; total yeast DNA was isolated by the method of Cryer et.al., (1975), and digested with the restriction endonucleases Eco RI and Cla I. Digested and undigested DNA fragments were separated by agarose gel electrophoresis and transferred to nitrocellulose filters by the method of Southern (Maniatis et.al., 1982).
  • Hybridization to the 1.25 kilo-base-pairs Sau 3A fragment of pET13:1, carrying the CUP-1 gene showed that the copper resistant ⁇ -galactosidase negative clones had a different hybridization pattern to that observed for brewing yeast transformants harbouring plasmid pEHB11.
  • the hybridization pattern obtained was indicative of the integration of the CUP-1 sequence of pEHB11 into the endogenous 2 ⁇ m plasmid of the brewing yeast strain.
  • the site of integration of the CUP-1 gene within the endogenous 2 ⁇ m plasmid of brewing yeast was shown to be within the region of DNA homology carried by the directly oriented DNA repeat sequences of pEHB11. This was determined by probing total DNA restriction digests with a 32 P labelled 2138 base-pair Eco RI - Hind III fragment derived from the plasmid pJDB110 (Beggs, 1981). This 2138 base-pair fragment harbours the origin of DNA replication of 2 ⁇ m B-form together with a single copy of the ⁇ inverted ⁇ repeat DNA sequence.
  • integration of CUP-1 within a region of DNA immediately adjacent to one of the inverted repeats results in a perturbation of the normal restriction pattern observed following hybridization to Southern transfers.
  • Standard restriction mapping coupled with Southern transfer and hybridization enabled the site of insertion to be localized within a 703 base-pair region of DNA bounded by an Eco RI site at coordinate O and an Xba I site at coordinate 703 of 2 ⁇ m B form (Broach 1981).
  • ⁇ integrants ⁇ The inheritable stability of the copper resistant phenotype in the copper resistant ⁇ -galactosidase negative clones of brewing yeast, hereinafter referred to as ⁇ integrants ⁇ , was analysed after growth under non-selective conditions.
  • Integrants were isolated and cultured in 10ml NEP containing 2% w/v glucose and 0.2mM CuSO 4 .7H 2 O to stationary phase. Yeast cells were then harvested by centrifugation and subcultured in fresh medium lacking copper sulphate. Yeast were grown to mid-exponential growth phase and then sub-cultured into fresh growth medium. The presence of the CUP-1 gene was monitored phenotypically by plating cells on NEP, 2% w/v glucose agar medium, followed by replica plating to the same medium supplemented with 0.5mM CuSO 4 .7H 2 O. This process of continuous growth under non-selective conditions was maintained for approximately 100-130 generations. The results presented in FIG.
  • Brewing yeast normally contains a single chromosomal copy of the CUP-1 gene located on a 5.2 kilo-base-pair Eco RI fragment.
  • Eco RI digested total yeast DNA with a 32 P labelled CUP-1 probe (e.g. 1.25 kilo-base-pair Sau 3A fragment from pET13:1, Henderson et.al., 1985).
  • Such a hybridization results in two bands of DNA homology corresponding to the 5.2 kilo-base-pair chromosomal CUP-1 fragment and a 3.0 kilo-base-pair fragment derived from the CUP-1 gene integrated into the endogenous 2 ⁇ m plasmid.
  • a comparison of the intensity of the two DNA bands by densitometric scanning of the exposed autoradiogram enables one to estimate the approximate copy number of the extrachromosomal CUP-1 gene relative to the number of chromosomal/genomic equivalents. In this way, it was estimated that the integrants possess approximately 87 copies of the extrachromosomal CUP-1 gene per CUP-1 genome equivalent.
  • Plasmid pEHB11 carries a unique KpnI restriction endonuclease site which is located within the directly oriented 703 base-pair homologous repeat, adjacent to the 3' terminus of the CUP-1 gene (FIG.1). This unique site can conveniently be used for the insertion of additional DNA sequences, for example a ⁇ gene of interest ⁇ , thereby enabling integration into the endogenous 2 ⁇ m plasmid of brewing yeast following successful transformation.
  • H$A Human Serum Albumin

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US5759802A (en) * 1988-10-26 1998-06-02 Tonen Corporation Production of human serum alubumin A
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US6545142B1 (en) 1988-11-11 2003-04-08 Medical Research Council Of The United Kingdom Single domain ligands, receptors comprising said ligands, methods for their production, and use of said ligands and receptors
US20080299618A1 (en) * 1988-11-11 2008-12-04 Medical Research Council Single domain ligands, receptors comprising said ligands, methods for their production and use of said ligands and receptors
US6248516B1 (en) 1988-11-11 2001-06-19 Medical Research Council Single domain ligands, receptors comprising said ligands methods for their production, and use of said ligands and receptors
US7306907B2 (en) 1988-11-11 2007-12-11 Cambridge Antibody Technology Limited Single domain ligands, receptors comprising said ligands, methods for their production, and use of said ligands and receptors
US20040110941A2 (en) * 1988-11-11 2004-06-10 Medical Research Council Single domain ligands, receptors comprising said ligands, methods for their production, and use of said ligands and receptors
US20030130496A1 (en) * 1988-11-11 2003-07-10 Medical Research Council Single domain ligands, receptors comprising said ligands, methods for their production, and use of said ligands and receptors
US20070207475A1 (en) * 1989-05-16 2007-09-06 Scripps Research Institute Method for producing polymers having a preselected activity
US8338107B2 (en) 1989-05-16 2012-12-25 Scripps Research Institute Method for producing polymers having a preselected activity
US7858359B2 (en) 1989-05-16 2010-12-28 Stratagene Method for tapping the immunological repertoire
US6680192B1 (en) 1989-05-16 2004-01-20 Scripps Research Institute Method for producing polymers having a preselected activity
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ZA874605B (en) 1988-03-30
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EP0251744A3 (en) 1988-06-22
AU614490B2 (en) 1991-09-05
GB8615701D0 (en) 1986-08-06

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